Synthesis and Evaluation of Chirally Defined Side Chain Variants of 7-Chloro-4-Aminoquinoline To Overcome Drug Resistance in Malaria Chemotherapy.

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

Broad spectrum anti-infective properties of benzisothiazolones and the parallels in their anti-bacterial and anti-fungal effects.

Various mono- and bis-benzisothiazolone derivatives were synthesized and screened against different strains of bacteria and fungi in order to understand the effect of multiple electrophilic sulfur atoms and substitution pattern in the immediate vicinity of reactive sulfur. Staphyllococcus aureus-ATCC 7000699, MRSA and S. aureus-ATCC 29213 (Quality Control strain) were more susceptible to this class of compounds, and the most potent derivative 1.15 had MIC50 of 0.4µg/mL (cf. Gentamicin=0.78µg/mL). CLogP value, optimally in the range of 2.5-3.5, appeared to contribute more to the activity than the steric and electronic effects of groups attached at nitrogen. By and large, their anti-fungal activities also followed a similar trend with respect to the structure and CLogP values. The best potency of IC50=0.1µg/mL was shown by N-benzyl derivative (1.7) against Aspergillus fumigatus; it was also potent against Candida albicans, Cryptococcus neoformans, Sporothrix schenckii, and Candida parapsilosis with IC50 values ranging from 0.4 to 1.3µg/mL. Preliminary studies also showed that this class of compounds have the ability to target malaria parasite with IC50 values in low micromolar range, and improvement of selectivity is possible through structure optimization.

Correlation between in vitro and in vivo antimalarial activity of compounds using CQ-sensitive and CQ-resistant strains of Plasmodium falciparum and CQ-resistant strain of P. yoelii.

Present efforts have been made to establish a correlation between in vitro and in vivo antimalarial activity using MIC, IC50 and IC90 values against CQ-sensitive (3D7) and CQ-resistant (K1) strains of Plasmodium falciparum and in vivo activity against Plasmodium yoelii. The method of discriminant function analysis (DFA) was applied to analyze the data. It was observed that in vitro IC90 values against both 3D7 and K1 strains (p < 0.001) have strong correlation with in vivo curative activity. The respective IC50 and IC90 values of compounds, which cured mice (i.e., animals did not show recrudescence of parasitemia even after 60 days posttreatment), ranged between 3 and 14 nM and 14 and 186 nM against 3D7 and between 9 and 65 nM and 24 and 359 nM against the K1 strain of P. falciparum. Whereas the IC50 and IC90 values of compounds which exhibited in vivo suppressive activity in mice ranged between 10 and 307 nm and 61 and >965 nM, respectively, against 3D7 and 75 and >806 nm and 241 and >1232 nM against the K1 strain of P. falciparum. The findings suggest that IC90 values against both 3D7 and K1 strains (p < 0.02) are the main contributors for the prediction of in vivo curative activity of a new molecule. Apart from this, a reasonable correlation between MIC and IC50 values of compounds has also been established.

Immunoprotective effect of lentinan in combination with miltefosine on Leishmania-infected J-774A.1 macrophages.

Rejuvenation of deteriorated host immune functions is imperative for successful annihilation of Leishmania parasites. The use of immunomodulatory agents may have several advantages as they conquer immunosuppression and, when given in combination, improve current therapeutic regimens. We herein investigated the immunostimulatory potency of a ß-glucan, lentinan either alone or in combination with short dose of standard drug, miltefosine on Leishmania-infected J-774A.1 macrophages. Our study shows that infected macrophages when stimulated with 2.5 µg/mL and above concentrations of lentinan secreted significant amount of host-protective molecules. The in vitro interaction between lentinan and miltefosine showed some synergy (mean sum of fractional inhibitory concentration [mean ∑FIC] 0.87) at IC50 level. Lentinan (2.5 µg/mL) plus low-dose miltefosine (2 µM) displayed heightened level of pro-inflammatory cytokines, IL-12 (13.6-fold) and TNF-α (6.8-fold) along with nitric oxide (7.2-fold higher) when compared with infected control. In combination group, we also observed remarkably (P<.001) suppressed levels of anti-inflammatory cytokines, IL-10 and TGF-ß, than that of untreated macrophages. Additionally, in comparison with infected group, we observed significant induction in phagocytic activity of macrophages in combination with treated group. Collectively, these findings emphasize the immunostimulatory effect of lentinan alone and in combination with low dose of miltefosine against Leishmania donovani.

Arteether resistance reversal by ketoconazole/fluconazole in rodent malaria parasite Plasmodium vinckei.

Artemisinin and its derivative arteether (ART) are fast acting antimalarial drugs against chloroquine-resistant. There are several partner drugs that are identified as a potential drug for artemisinin combination therapy (ACT) to develop as the antimalarial drug. Limited studies have been carried out in ART drug combination that may have more promising as ACT for resistant Plasmodium parasite. Here, we are the first to show the ART drug resistance reversal in Plasmodium vinckei by using antifungal azole compounds ketoconazole (KTZ) and fluconazole (FCZ). Our previous study has shown that higher antioxidant enzyme, glutathione, and less hemozoin may be correlated with ART resistance in P. vinckei (PvAR). We further hypothesized that glutathione and heme catabolism may be interfered by KTZ and FCZ, resulting in an increased efficacy of ART in PvAR parasite. The results of present study demonstrate synergetic effect of KTZ and FCZ against PvAR parasite, since none of the mice developed infection up to day 10 after combination with ART. These results further showed that ED90 of ART was reduced from 17.23 to 2.19 and 2.56 mg/kg when used in combination with KTZ and FCZ, respectively. Resultant, activity enhancement index (AEI) of ART is significantly increased to 8.60 and 6.73 with partner agents. These studies propose the possibility of ART drug combination that may be helpful in prolonging the life of drug and a promising lead to reduce the chance of resistance development of artemisinin and its derivative.

A natural product inspired hybrid approach towards the synthesis of novel pentamidine based scaffolds as potential anti-parasitic agents.

A natural product inspired molecular hybridization approach led us to a series of novel pentamidine based pyrimidine and chalcone scaffolds. All the hybrids were evaluated for their anti-leishmanial potential. Most of the screened compounds have showed significant in vitro anti-leishmanial activity with less cytotoxicity in comparison to the standard drugs (pentamidine, sodium stibogluconate, and miltefosine). Additionally, anti-malarial screening of these compounds was also done and four compounds have shown superior activity against chloroquine resistance strain (K1) of Plasmodium falciparum.

Coumarin-trioxane hybrids: synthesis and evaluation as a new class of antimalarial scaffolds.

First synthesis of novel coumarin-trioxane hybrids is reported. The synthesis was achieved via condensation of ß-hydroxyhydroperoxides with coumarinic-aldehydes in presence of p-toluenesulfonic acid in good yields and the novel hybrids were evaluated for their antimalarial activity both in vitro and in vivo.

In vitro activities of new 2-substituted quinolines against Leishmania donovani.

A series of 9 quinolines and 18 styrylquinolines was evaluated for the drugs' in vitro antileishmanial activities and cytotoxicities. The 7-aroylstyrylquinoline scaffold appeared to be the most promising one, with the most interesting compound, no. 35, exhibiting a 50% inhibitory concentration (IC(50)) of 1.2 µM and a selectivity index value of 121.5. Compound 35 was 10-fold and 8-fold more active than miltefosine and sitamaquine, the reference compounds, with selectivity indexes 607-fold and 60-fold higher, respectively.

Sterile protection of monkeys against malaria after administration of interleukin-12.

An estimated 300-500 million new infections and 1.5-2.7 million deaths attributed to malaria occur annually in the developing world, and every year tens of millions of travelers from countries where malaria is not transmitted visit countries with malaria. Because the parasites that cause malaria have developed resistance to many antimalarial drugs, new methods for prevention are required. Intraperitoneal injection into mice of one dose of 150 ng (approximately 7.5 micrograms per kg body weight) recombinant mouse interleukin-12 (rmIL-12) 2 days before challenge with Plasmodium yoelii sporozoites protects 100% of mice against malaria. We report that one subcutaneous injection of 10 micrograms/kg recombinant human IL-12 (rhIL-12) 2 days before challenge with P. cynomolgi sporozoites protected seven of seven rhesus monkeys. Protection was associated with marked increases in plasma levels of interferon-gamma (IFN-gamma), and relative increases of lymphoid cell messenger RNA coding for IFN-gamma and several other cytokines. We speculate that rIL-12 protects monkeys through IFN-gamma-dependent elimination of P. cynomolgi-infected hepatocytes. This first report of rIL-12-induced protection of primates against an infectious agent supports assessment of rhIL-12 for immunoprophylaxis of human malaria.

In-vitro culture of Plasmodium falciparum: utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay.

Studies were carried out to establish the potential of RPNI medium for drug-sensitivity studies using the MSF assay. The drug sensitivity of standard anti-malarials was compared using both the ((3)H) Hypoxanthine incorporation assay and the MSF assay. The media supplements used during the study have been human serum, FBS and ALBUMAX-II. Drug sensitivity of two parasite lines, adapted to grow separately in conventional as well as in RPNI medium was compared to observe the effect of RPNI medium on functional characteristics of the parasite. The results revealed identical IC(50) values of standard anti-malarials obtained by both the ((3)H) Hypoxanthine incorporation assay and the MSF assay and no untoward effect of FBS and ALBUMAX-II could be noticed on the chemo-sensitivity of standard anti-malarials. Apart from this the chemo-sensitive response of parasite line adapted to grow in RPNI medium was observed to be intact. These findings showed that RPNI medium has potential to be used for chemo-sensitivity studies and the MSF assay being more convenient was observed to be most suitable assay for bio evaluation of new molecules.

Plasmodium vinckei: infectivity of arteether-sensitive and arteether-resistant parasites in different strains of mice.

Malaria is one of the most lethal parasitic infections in the world. The lethality of the parasite depends on the rate of multiplication of the parasite within host erythrocytes. Different strains of the malaria parasite often respond in a different way to the same strain of mice or vice versa. In the present study, we investigated the course of infection of the arteether-sensitive and arteether-resistant Plasmodium vinckei parasites in Swiss albino AKR (inbred) and AJ (outbred) mice. The higher parasite burden and mortality were observed in the sensitive parasite-infected mice, whereas the infection with the resistant parasite was non-lethal. Resistant parasite-infected mice developed a moderate level of parasitemia that decreased gradually throughout the infection. The microscopic examination suggests that the resistant parasite invades reticulocytes more efficiently than normocytes, regardless of the mouse strain examined. Since the reticulocytes are rare in blood circulation, it limits the increase in parasite proliferations, while arteether-sensitive parasites can invade both mature normocytes and reticulocytes, resulting in the mortality of the mice. However, treatment with phenylhydrazine in Swiss mice results in reticulocytosis, which transforms the non-lethal resistant parasites to produce lethal infections. Our findings demonstrate that the characteristic response during infections with the arteether-resistant strain is dependent on the availability of reticulocytes in peripheral blood circulation. We can use this model for identifying the interaction between host and artemisinin derivative-resistant parasites.

Synthesis of new 4-aminoquinolines and quinoline-acridine hybrids as antimalarial agents.

Despite emergence of resistance to CQ and other 4-aminoquinoline drugs in most of the endemic regions, research findings provide considerable support that there is still significant potential to discover new affordable, safe, and efficacious 4-aminoquinoline antimalarials. In present study, new side chain modified 4-aminoquinoline derivatives and quinoline-acridine hybrids were synthesized and evaluated in vitro against NF 54 strain of Plasmodium falciparum. Among the evaluated compounds, compound 17 (MIC=0.125 µg/mL) was equipotent to standard drug CQ (MIC=0.125 µg/mL) and compound 21 (MIC=0.031 µg/mL) was four times more potent than CQ. Compound 17 showed the curative response to all the treated swiss mice infected with CQ-resistant N-67 strain of Plasmodium yoelii at the doses 50 mg/kg and 25 mg/kg for four days by intraperitoneal route and was found to be orally active at the dose of 100 mg/kg for four days. The promising antimalarial potency of compound 17 highlights the significance of exploring the privileged 4-aminoquinoline class for new antimalarials.

Material-Microbe Interfaces for Solar-Driven CO2 Bioelectrosynthesis.

Sustainable production of solar-based chemicals is possible by mimicking the natural photosynthetic mechanism. To realize the full potential of solar-to-chemical production, the artificial means of photosynthesis and the biological approach should complement each other. The recently developed hybrid microbe-metal interface combines an inorganic, semiconducting light-harvester material with efficient and simple microorganisms, resulting in a novel metal-microbe interface that helps the microbes to capture energy directly from sunlight. This solar energy is then used for sustainable biosynthesis of chemicals from CO2. This review discusses various approaches to improve the electron uptake by microbes at the bioinorganic interface, especially self-photosensitized microbial systems and integrated water splitting biosynthetic systems, with emphasis on CO2 bioelectrosynthesis.

Synthesis of 9-anilinoacridine triazines as new class of hybrid antimalarial agents.

There is challenge and urgency to synthesize cost-effective chemotherapeutic agents for treatment of malaria after the widespread development of resistance to CQ. In the present study, we synthesized a new series of hybrid 9-anilinoacridine triazines using the cheap chemicals 6,9-dichloro-2-methoxy acridine and cyanuric chloride. The series of new hybrid 9-anilinoacridine triazines were evaluated in vitro for their antimalarial activity against CQ-sensitive 3D7 strain of Plasmodium falciparum and their cytotoxicity were determined on VERO cell line. Of the evaluated compounds, two compounds 17 (IC(50)=4.21 nM) and 22 (IC(50)=4.27 nM) displayed two times higher potency than CQ (IC(50)=8.15 nM). Most of the compounds showed fairly high selectivity index. The compounds 13 and 29 displayed >96.59% and 98.73% suppression, respectively, orally against N-67 strain of Plasmodium yoelii in swiss mice at dose 100 mg/kg for four days.

Synthesis of novel thiourea, thiazolidinedione and thioparabanic acid derivatives of 4-aminoquinoline as potent antimalarials.

In search of new 4-aminoquinolines which are not recognized by CQR mechanism, thiourea, thiazolidinedione and thioparabanic acid derivatives of 4-aminoquinoline were synthesized and screened for their antimalarial activities. Thiourea derivative 3 found to be the most active against CQ sensitive strain 3D7 of Plasmodium falciparum in an in vitro model with an IC(50) of 6.07ng/mL and also showed an in vivo suppression of 99.27% on day 4 against CQ resistant strain N-67 of Plasmodium yoelii.

Search for new pharmacophores for antimalarial activity. Part II: synthesis and antimalarial activity of new 6-ureido-4-anilinoquinazolines.

Synthesis of new 6-ureido-4-anilinoquinazolines have been accomplished and their in vitro antimalarial activity against chloroquine-sensitive P. falciparum have been examined. Out of 64 compounds evaluated, the IC(50) of 16 compounds which have displayed MIC of 0.25 microg/mL were also recorded. One of the compounds (24 g) had IC(50) value of 2.27 ng/mL which was equipotent to the standard drug chloroquine used in the bioassay. The in vivo evaluation of a few compounds among the series led to discovery of one analog (30 g) displaying 40% curative activity (28 days) against mdr P. yoeillinigeriensis at an oral dose of 100 mg/kg x 4 days.

Synthesis of oxalamide and triazine derivatives as a novel class of hybrid 4-aminoquinoline with potent antiplasmodial activity.

Frequency of malaria and its resistance to chemotherapeutic options are emerging rapidly. To counter this problem, a series of 4-aminoquinolines having oxalamide and triazine functionalities in the side chain were synthesized and screened for their antimalarial activities. Triazine derivative 48 found to be the most active against CQ sensitive strain 3D7 of Plasmodium falciparum in an in vitro assay with an IC(50) of 5.23 ng/mL and oxalamide derivative 13 showed an in vivo suppression of 70.45% on day 4 against CQ resistant strain N-67 of Plasmodium yoelii.

Search for new pharmacophores for antimalarial activity. Part I: synthesis and antimalarial activity of new 2-methyl-6-ureido-4-quinolinamides.

A total of 80 new 2-methyl-6-ureido-4-quinolinamides were synthesized and evaluated for their antimalarial activity. Several analogs elicited the antimalarial effect at MIC of 0.25 mg/mL against the chlooquine-sensitive P. falciparum strain. The IC(50) values of the active compounds were observed to be in ng/mL range and two of the analogs have better IC(50) value than the standard chloroquine. In the in vivo assay against mdr CQ resistant P. yoelii N67/P. yoelii nigeriensis, however, none of the compound showed complete suppression of parasitemia on day 7. One of the compounds displayed significant antibacterial effect against several strains of bacteria and was many-fold better than the standard drug gentamicin.

Photosensitization of electro-active microbes for solar assisted carbon dioxide transformation.

Tandem bio-inorganic platform by combining efficient light harvesting properties of nano-inorganic semiconductor cadmium sulfide (CdS) with biocatalytic ability of electro-active bacteria (EAB) towards carbon dioxide (CO2) conversion is reported. Sulfur was obtained from either cysteine (EAB-Cys-CdS) or hydrogen sulfide (EAB-H2S-CdS) and experiments were carried out under similar conditions. Anchoring of the nano CdS cluster on the microbe surface was confirmed using electronic microscope. Bio-inorganic hybrid system was able to produce single and multi-carbon compounds from CO2 in visible spectrum (λ > 400 nm). Though, acetic acid was dominant (EAB-Cys-CdS, 1.46 g/l and EAB-H2S-CdS, 1.55 g/l) in both the microbe-CdS hybrids, its concentration as well as product slate varied significantly. EAB-H2S-CdS produced hexanoic acid and less methanol fraction, while the EAB-Cys-CdS produced no hexanoic acid along with almost double the concentration of methanol. Due to easy harvesting process, this bio-inorganic hybrid represents unique sustainable approach for solar-to-chemical production via CO2 transformation.

Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients correlates with the progression of disease.

Free radicals are implicated in numerous disease processes including motor neuron degeneration (MND). Antioxidant defense enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the erythrocytes are capable of detoxifying reactive oxygen species produced endogenously or exogenously. In the present study, the extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 sporadic amyotrophic lateral sclerosis (ALS) patients and 20 controls. We observed that lipid peroxidation in the erythrocytes of amyotrophic lateral sclerosis patients significantly increased with respect to controls (P<0.001). On the other hand, catalase activity was found to be significantly lower (P<0.001). The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione levels were also found to be significantly reduced in ALS patients compared to healthy subjects (P<0.001, P<0.01 and P<0.01, respectively). It was further observed that lipid peroxidation started to increase and catalase, glutathione reductase, glucose-6-phosphate dehydrogenase enzyme activities and glutathione levels started to decrease as amyotrophic lateral sclerosis progressed from 6 to 24 months, suggesting a correlation between these parameters and duration of amyotrophic lateral sclerosis. This study confirms the involvement of oxidative stress during the progression of amyotrophic lateral sclerosis and the need to develop specific peripheral biomarkers.